Biochemistry and biological function of mammalian Dnmt1

DNA甲基化是哺乳动物基因调控的重要过程。到目前为止，在哺乳动物中发现了三种活跃的DNA甲基转移酶：Dnmt1参与维持甲基化；Dnmt3a和3b参与从头DNA甲基化。Dnmt1是一个包含1620个氨基酸残基的大蛋白。在胚胎发生过程中，完全敲除Dnmt1酶是致命的。Dnmt1数量的减少导致体内DNA的去甲基化。此外，它还能阻止DNA复制并诱导细胞凋亡。这些影响是否由于DNA的去甲基化尚不清楚。Dnmt1在体外被磷酸化并与其他几种蛋白相互作用。生化数据表明，它也可能涉及一个称为甲基化扩散的过程，这在肿瘤进展过程中经常观察到。在本应用中，我们打算制备携带单个或少量氨基酸交换的Dnmt1变异体，专门处理蛋白质的定义生化功能，并在体外表征突变酶。随后将产生内源性dnmt1基因中含有相同定义交换的转基因小鼠，以研究dnmt1某些特性在体内的功能。采用这种组合方法，我们试图研究a) Dnmt1催化活性的丧失是否导致完全敲除的胚胎致死，b) Dnmt1与PCNA蛋白的相互作用是否在体内发挥作用。c)在体外观察到的Dnmt1的磷酸化是否与体内相关，d)在体外观察到的Dnmt1对未修饰DNA的甲基化的变构激活是否在体内有作用，并且可能导致甲基化的扩散。到目前为止，这种结合定点诱变和基因靶向的实验还没有使用任何DNA甲基转移酶进行。最后，我们试图研究另一种DNA甲基转移酶Dnmt2的生物学作用，Dnmt2的功能迄今为止尚不清楚，通过制备小鼠敲除菌株并仔细研究转基因动物的几代。我们期望这些实验的结果将大大促进我们对DNA甲基化和表观遗传基因控制的认识。

DNA methylation is an essential process in mammals involved in several instances of gene regulation. So far, three active DNA methyltransferases have been found in mammals: Dnmt1 involved in maintenance methylation; Dnmt3a and 3b involved in de novo DNA methylation. Dnmt1 is a large protein comprising 1620 amino acid residues. Complete knock-out of the Dnmt1 enzyme is lethal during embryogenesis. Reduction of the amount of Dnmt1 causes demethylation of the DNA in vivo. Furthermore, it leads to an arrest of DNA replication and induces apoptosis. If these effects are due to demethylation of the DNA is not known. Dnmt1 is phosphorylated and interacts with several other proteins in vitro. Biochemical data suggests that it might also be implicated in a process called spreading of methylation, which is often observed in the course of tumor progression. In this application we intend to prepare Dnmt1 variants carrying single or few amino acid exchanges that specifically address defined biochemical functions of the protein and characterize the mutant enzymes in vitro. Subsequently transgenic mice will be generated which contain the same defined exchanges in their endogenous dnmt1 gene to investigate the in vivo function of certain properties of Dnmt1. Following this combined approach, we attempt to investigate a) if the loss of catalytic activity of Dnmt1 is responsible for embryonic lethality of the full knock-out, b) if the interaction of Dnmt1 with the PCNA protein has a role in vivo, c) if phosphorylation of Dnmt1 that has been observed in vitro is of relevance in vivo and d) if an allosteric activation of Dnmt1 for methylation of unmodified DNA that has been observed in vitro and might cause spreading of methylation has a function in vivo. Such experiments which combine site-directed mutagenesis and gene targeting, so far, have not been carried out with any DNA methyltransferase. Finally, we attempt to investigate the biological role of an additional DNA methyltransferase, Dnmt2, whose function so far is not known by preparing a mouse knock-out strain and carefully investigate the transgenic animals over several generations. We expect that the results of these experiments will considerably further our knowledge on DNA methylation and epigenetic gene control.